As generally known, platelet rich plasma (PRP) is an autologous material separated from whole blood through density gradient centrifugation, and is an inactive substance that includes a large amount of platelets concentrated with respect to a small amount of plasma, and contains leukocytes in a high-concentration.
Inactivated platelets within a blood vessel maintain a round shape while circulating through the blood vessel. It is known that the platelets have a life span of about 10 days, and are present in an amount of about 2˜108 with respect to 1 mL of the blood.
Platelets within PRP are activated through substances included within a blood vessel, and then release growth factors and various active substances. The activating substances include collagen, thrombin, adenosine diphosphate (ADP) and epinephrine. Especially, collagen and thrombin are known as strong agonists. It is known that collagen induces the activation of the platelets through the adhesion of its specific sequence onto the platelets as shown in FIG. 1.
When activating factors activate the platelets, the platelets release growth factors due to degranulation of their alpha granules. The factors perform an important role in initial wound healing. Herein, the released growth factors include platelet derived growth factor (PDGF-AB), transforming growth factor-b1 (TGF-b1), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), insulin-like growth factor (IGF) and the like. Also, PRP releases cytokine, etc. under in vitro condition, thereby synthesizing DNA of fibroblasts. This increases the production of collagen, and thus allows a collagen structure to be organized.
However, in the application of PRP in a liquid state, it is difficult to inject the PRP into a wound, and there exists a loss of surrounding tissues. Thus, it is required to develop clotting gelation accompanied by physical properties. For the gelation, research on the use of thrombin has been recently mainly conducted. However, there is a report that in the use of thrombin (mainly bovine-derived), that is an animal-derived protein, an immune reaction such as lupus was observed. In other words, thrombin is known as a factor having clinical problems related to an antibody. Thrombin is the most potent component as a platelet activator. However, when thrombin is used, antibodies to thrombin, prothrombin, factor V, and cardiolipin are required. Also, through animal research, it was found that there are clinical problems such as after-operation bleeding, and an autoimmune syndrome. Although these problems rarely occur, they cannot be neglected in development. The use of thrombin may cause the spread of damaged wound, abnormal strength of gel or the like.
Furthermore, due to high contractility of a thrombin-activating gel, there exists a difficulty in the surgical procedure for filling a wound space. Thus, the importance of a replacement material for thrombin has been recognized.
Collagen is a rigid fibrous protein, and is a main protein component of a mammalian connective tissue. It makes up 30% or more of total proteins. The collagen provides shape, strength and flexibility to the tissue, and has various functions such as tissue scaffolding, cell adhesion, cell migration, blood vessel production, tissue morphogenesis, tissue repair, and the like. Collagen, as a strong agonist of platelets, activates the platelets, and causes platelet aggregation. Fibrillar collagen more strongly induces platelet aggregation and supports greater platelet adhesion than a soluble collagen. Although the reason for such a difference has not been clearly proven, it is assumed that there is a possibility that the fibrillar collagen binds to a molecule increasing the action of activated platelets.
Type I collagen makes up a majority of mammalian connective tissue. Also, as a natural scaffold, it is most actively researched in the fields of regenerative medicine and tissue engineering. Due to these advantages of type I collagen, the type I collagen performs a role of activating platelets by replacing thrombin, and maintaining the shape.
However, there has been no composition for inducing tissue regeneration by activating PRP with a calcium chloride solution and the type I collagen. In other words, there has been no substitute for thrombin. Thus, only the thrombin (mainly bovine-derived), that is, an animal-derived protein, has been used despite its many problems.